The present invention provides a new surgical procedure of partial hepatectomy, in nonhuman primates. More particularly, the present invention provides an open biopsy procedure, alternating non-anatomic and anatomic partial liver resection on a liver lobe.
The first relevant anatomic studies of the liver emerged with the endeavors of Herophilus and Erasistratus between 310 and 280 B.C. However, it was not until after the development of anesthesia and antisepsis that the first formal hepatic resections were performed in human medicine during late 1800""s. After a burst of pioneering activity from 1880 to 1910 (1, 2), little progress was made until World War II. In the last 50 years, remarkable advances in the surgical techniques of human liver resections were made (3, 4, 5) based on the increasing knowledge of the anatomy of the liver. Accurate and safe surgical procedures have been implemented to intervene at the level of the liver parenchyma and bile ducts with increased safety margin and optimal clinical outcomes.
Hepatic resection, or hepatectomy, is defined as the removal of a liver segment or a complete liver lobe. Partial hepatic tissue resection is immediately followed by the regeneration of the liver parenchyma within a few weeks (6, 7, 8, 9, 10, 11, 12). The regenerative response of the liver is proportional the amount of tissue removed leading eventually to the restoration of the original size and with adaptation to whole body size if liver from large animals are transplanted into small animals of the same species.
In human surgery, hepatectomies are classified as total when the organ is used for liver transplantation and partial when, e.g., areas of isolated primary or secondary metastasis are removed. Partial hepatectomies are subdivided in anatomic and non-anatomic resections. Anatomic resections are defined by the vascular structure, while non-anatomic resections are performed at the level of the liver parenchyma. At least six different techniques for human hepatectomy have been described in the literature (6).
In veterinary medicine and research, liver biopsy is an important diagnostic tool in evaluating hepatic morphology, assessing molecular and biochemical properties of the liver for both clinical and research purposes. Indications for partial liver removal include localized masses such as abscesses or neoplasia, trauma, vascular alterations, viral hepatitis, and research. The techniques available for partial liver resection can be classified into two major groups: open and closed liver biopsies (13).
The closed biopsy procedures are performed by percutaneous needle biopsy using a transabdominal or transthoracic approach. Closed liver biopsy can be performed rapidly under local anesthesia and is best suited for multisampling. However, it is limited by the small sample size (about 100 mg) and numerous complications such as puncture of the gallbladder or adjacent organs and bacterial peritonitis that can occur due to the blinded fashion by which this technique is performed (13, 14, 15, 16). A better accuracy in sample collection can be achieved by combining a keyhole technique to needle biopsy puncture (13, 14). In this semi open technique, a cranial ventral midline incision is performed to allow localization and mobilization of the liver lobes by direct digital palpation. However, even if the risk of aberrant puncture can be decreased, this technique offers the disadvantage of being still limited by its small sample size.
Open techniques for partial liver resection have been previously described in rats, dogs and nonhuman primates (13, 17, 18, 19, 20, 21, 22, 23). Following the terminology used in human liver surgery, open biopsies in animals are used to perform partial non-anatomic liver resections. These techniques can be categorized into four major groups: punch biopsy, wedge resection, finger fractures, and ligature fractures.
The punch biopsy uses Keys cutaneous biopsy punch for collecting of small, superficial samples in essentially any location on the liver surface (13).
Wedge liver resection can be used to remove larger areas located on the edge of the liver lobes. However, this technique involves the application of a horizontal mattress, using interlapping sutures along the line that will give the desired piece of liver. This procedure is invasive but allows the dissection of relatively large liver samples (50-100 grams). The potential risk of developing post-surgery intrahepatic hematomas due to the significant amount of suture material left deep with the hepatic parenchyma has been underlined (13, 21).
One of the most commonly used procedures for partial lobectomy of large sections of the liver is the finger fracture technique. In this technique, the portion of hepatic tissue to be removed is fractured by the fingers and the difference in resistance between the liver parenchyma and the intrahepatic ducts and vessels is readily apparent, thus making them easily identified. Once the hepatic segment has been crushed away from the ducts and vessels, they are clamped, ligated and severed controlling thereby hemorrhage and bile leakage (13, 15, 18, 20, 24). However, in rhesus macaques studies have shown that extensive blunt dissection causes the release of proteolytic enzymes into the circulation resulting in postoperative P02 depression (20).
The ligature fracture technique has been described has the quickest and most simple method to collect large biopsy liver samples (15, 23). In both African green and cynomolgus monkeys, the ligature technique was used to obtain multiple large (2 to 4 gr) liver biopsy samples. This technique was applied 195 times in 84 animals using three different surgical approaches indicating that 2 to 3 partial liver resections can be performed on the same animal. However, data indicated that serum alanine aminotransferase (ALT) concentration undergoes a transient but significant elevation in association with acute hepatocellular damage. Nolan and Conti (21) described a procedure for removing large (10-20 gr) liver samples in chimpanzees using an automatic stapling device for cell culture experiment purposes. Three biopsies were performed on the same animal but from areas not involved in the previous biopsy.
However none of these studies addressed a suitable method to perform multiple (more than 3) liver biopsies in nonhuman primates in which approximately 20 gr of tissue would be removed in each instance for tissue slices and cell culture purposes. In addition, a procedure allowing more than one intervention on the same liver lobe has not been described.
The 3R""s principle (refining, reducing, replacing) is one of the most important and leading issue in Laboratory Animal Science. In vitro testing is a fundamental tool to optimize, reduce, refine, and replace the use of laboratory animals, particularly working with nonhuman primates.
An objective of this invention is to provide a new surgical procedure of partial hepatectomy in the nonhuman primate allowing repeated removing of liver tissue samples from the same animal for the preparation of liver tissue slices and cell culture of hepatocytes.
The new surgical procedure of partial hepatectomy in the nonhuman primate described in this invention allows repeated removal of large (about 20 gram) liver tissue samples from a single animal for the preparation of liver tissue slices and primary cell culture of hepatocytes. This open biopsy procedure, alternating non-anatomic and anatomic partial liver resection monthly on the same liver lobe, follows a surgical protocol allowing animal welfare, survival, and multiple sampling (at least 8) from the same animal. This procedure for multiple partial liver resection thus reduces considerably the number of animals needed for research purposes. This new surgical procedure is simple and safe. It does not produce pain, uncontrollable hemorrhage, anemia, bile leakage into the peritoneal cavity, dehydration, or hepatic insufficiency.
In more detail, the procedure developed and described herein preferably consists in the application of alternative non-anatomic and anatomic hepatectomy resection for eight removals of liver tissue sections from the same animal. Each operation is performed monthly on the same animal. The procedure consists in eight surgery steps alternating liver lobe clamping and resection of a segment of parenchyma without the use of ligature fracture followed after one month by an intervention on the same liver lobe clamping the vascular pedoncule for total lobectomy. These two alternative steps were repeated on the left lateral, right lateral, left central, and right central liver lobes, respectively.
Thus, the present invention provides a surgical procedure of partial hepatectomy in a nonhuman primate, which comprises repeated removal from the same animal of large liver tissue samples suitable for the preparation of liver tissue slices and primary cell culture of hepatocytes.
The surgical procedure herein preferably follows an open biopsy procedure following a protocol alternating non-anatomic and anatomic partial hepatic resection for eight removals of liver tissue sections from the same animal.
A particularly preferred embodiment of the surgical procedure of the present invention comprises the sequential steps of, (A) on the left lateral liver lobe, clamping and resectioning a segment of parenchyma without the use of ligature fracture followed after one month by resection of the remaining liver lobe after clamping and ligation of the vascular pedoncule, (B) on the right lateral liver lobe, clamping and resectioning a segment of parenchyma without the use of ligature fracture followed after one month by resection of the remaining liver lobe after clamping and ligation of the vascular pedoncule, (C) on the left central liver lobe, clamping and resectioning a segment of parenchyma without the use of ligature fracture followed after one month by resection of the remaining liver lobe after clamping and ligation of the vascular pedoncule, and (D) on the right central liver lobe, clamping and resectioning a segment of parenchyma without the use of ligature fracture followed after one month by resection of the remaining liver lobe after clamping and ligation of the vascular pedoncule.
Thus this invention provides a method of promoting animal welfare, survival, and reduction in the number of animals employed for research purposes on liver tissue slices and primary culture of human hepatocytes.